The present invention relates to a monoclonal antibody which is specifically reactive with a ribonucleotide reductase R2 subunit and which inhibits ribonucleotide reductase activity, as well as a hybridoma cell line producing the monoclonal antibody. The present invention further relates to a method of immunologically detecting a ribonucleotide reductase R2 subunit using the monoclonal antibody.
A ribonucleotide reductase (hereinafter referred to as "RNR") is an enzyme catalyzing the reduction of ribonucleoside diphosphate into deoxyribonucleoside diphosphate which is a part in DNA. Since RNR activity is strongly correlated with a cell proliferation rate and since deoxyribonucleotide is reluctant to pool in cells, RNR controls a rate-determining step of DNA synthesis (Annu. Rev. Biochem., 48, 133-158, 1979), and can be considered as a cell cycle marker and a proliferation marker. In fact, it is known that RNR activity increases or decreases depending on the cell cycle, and that the level of RNR activity is lower at G0/G1 phase and is the highest at S phase. RNR is composed of two subunits R1 and R2, either of which does not individually have any activity at all. It is reported that since the R1 subunit is always expressed constantly and sufficiently, RNR activity is controlled by the amount of the R2 subunit expressed, which amount depends on the cell cycle J.B.C., 256 (18), 9436-9440, 1981!. It is further reported that the level of RNR activity is higher in human tumor tissue than in human normal tissue (Life Science, 28, 1007-1014, 1981), and that RNR can be utilized as a tumor marker and a tumor proliferation marker.
A conventional method of determining RNR activity (Analytical Biochemistry, 34, 123-130, 1970) is not satisfactory because of the difficulty in determining the activity of RNR scattered in a tissue. A simple and accurate method of detecting RNR has been in demand.
An anti-RNR monoclonal antibody which is reactive with an R1 subunit and which inhibits RNR activity has been known Acta Chem. Scand., B36 (5), 343, 1982!. A monoclonal antibody which is reactive with an R2 subunit has been known The EMBO Journal, 7 (6), 1615, 1988)!. But the known monoclonal antibody which is reactive with R2 subunit does not inhibit RNR activity.
An object of the present invention is to provide a monoclonal antibody which is specifically reactive with an R2 subunit of human RNR, and which inhibits RNR activity.
The monoclonal antibody of the present invention is a neutral antibody against RNR, and thus it inhibits the RNR activity dependent on the concentrations of the monoclonal antibody.
By Western blotting and immunoprecipitation using the present monoclonal antibody, it is found that the present monoclonal antibody is reactive with a protein having a molecular weight of 45K daltons which coincides with the molecular weight of the R2 subunit of RNR. Further, by the immunological staining of culture cells and various human tissues using the present monoclonal antibody, it is found that the monoclonal antibody can specifically detect RNR in the cells and human tissues.
The monoclonal antibody of the present invention is useful not only for immunologically detecting a R2 subunit of RNR in tissues and cells, but also for studying the biological characterization of RNR. Thus, it is expected that the fundamental study of cancer will be promoted by analysis of RNR.